The green fluorescent protein (GFP) from jellyfish Aequorea victoria and its fluorescent homologues from Anthozoa corals have become invaluable tools for in vivo imaging of cells and tissues. Anthozoa GFP-like proteins are available in colors and with features unlike those of GFP variants and, thus, provide powerful templates for new probes for molecular labeling and intracellular detection. Several Anthozoa GFP-like proteins have been already developed into biotechnological tools. However, their photochemical and oligomeric properties limit their usefulness as molecular probes. Our analysis of chromophore formation mechanisms and color determinants suggest that monomeric proteins with novel spectral and photochemical features can be designed. On the basis of existing and novel monomeric red-shifted fluorescent proteins and chromoproteins, we plan to develop two types of protein labels, complementary to the existing GFP tools. These include Aim 1: monomeric fluorescent timers that change fluorescent color with time, and Aim 2: photoactivatable fluorescent proteins, which are originally dark but become fluorescent upon irradiation with violet light. We will also develop molecular evolution techniques consisting of rational, combinatory and random mutagenesis of candidate proteins followed by extensive flow cytometry and multiwell plate spectrometer screening. We will correlate the mutagenesis process with spectral and photochemical changes to gain insight into the molecular evolution of chromophore structures responsible for fluorescence properties and apply these to the next rounds of mutagenesis. In Aim 3, the fluorescent variants will be thoroughly characterized in mammalian cells as fusion tags, and the advanced probes will be utilized in trafficking and endocytosis studies of the human dopamine transporter. Elucidation of how its cell surface expression is regulated will enhance understanding of normal neurotransmission, as well as brain diseases like drug addiction. The anticipated end result of the proposed research is a set of molecular fluorescent probes that will be as versatile as GFP tools. These probes will expand the GFP-technology to allow simultaneous detection of lifetime, dynamics and interaction of several proteins in a single cell. This, in turn, will lead to development of new quantitative methods and applications of a kinetic microscopy of living cells.